The objective of this program is to understand the molecular basis of conservative site-specific recombination by the lambda integrase family of recombinases. This family of about 30 recombinases from bacteria and yeast mediates strand exchanges between two sequence-specific DNA substrates. The natural functions of these genetic rearrangements include the integration and excision of phage genomes and the maintenance of circular replicons. In recent years, these recombination systems have found an increasing role in the construction of large cloning vehicles and as key components of powerful genetic manipulations in plant and mammalian cells. Their potential as tools in genetic approaches to understanding and curing disease is enormous. The proposed work focuses on the Cre/lox system of bacteriophage P1 as a prototype recombination system from the integrase family. The Cre/lox system operates using a single recombinase (Cre) and a 34-base pair DNA target (lox). This program involves a structural approach to understanding Cre/lox recombination on a molecular level. High resolution X-ray crystallography of Cre/lox complexes will be used to establish (i) the structure of Cre recombinase, (ii) the basis of DNA- binding specificity and active site formation in the Cre/DNA interface, and (iii) an architectural framework for understanding site-specific recombination in the integrase family. The resulting stereochemical models will be related to existing biochemical and genetic data and mechanistic inferences will be tested experimentally. The long-term objective of this work is to engineer Cre recombinase and other site-specific recombinases to perform with altered specificity, directionality, and catalytic efficiency. Because these systems have already assumed prominent roles in the construction of genetic models for understanding the consequences of specific gene expression in mice, the proposed work is of fundamental biomedical importance.